Coffee is prepared from the roasted beans of the plants of the genus Coffea, generally from the species C. arabica. Beans are the seeds of the coffee plant and are obtained by processing the fruit, most ideally mature fruit which commands the best price due to its superior quality. In the past, high quality "gourmet" coffee was hand picked. This is necessary because the fruits of a coffee tree do not ripen uniformly and thus there are both mature and immature fruit on the same tree. In the past, this was not a serious problem as most coffee is grown in areas of the world where labor is plentiful and not expensive. However, more recently lack of abundant and inexpensive labor has become a major contributor to decreased productivity in coffee production. To increase productivity some regions of the world, such as the largest coffee producing country, Brazil, have resorted to strip harvesting where workers rapidly remove all fruit from a branch whether ripe or unripe. This increases the speed of harvesting but decreases the yield of the highest quality beans as much of the fruit is immature (green).
Furthermore, the lack of uniform ripening has seriously limited the effectiveness of mechanical harvesting. The force required to remove mature fruit (cherry) from the tree is similar to the force required to remove green fruit. Thus, mechanical harvesters do not distinguish well between green and cherry and a large amount of immature fruit is harvested along with mature fruit. This greatly decreases the yield of mature fruit and limits productivity. If coffee fruit ripening could be controlled so that all fruit ripened at one time, both the strip method of hand harvesting and mechanical harvesting would be much more efficient and a higher percentage of the harvested fruit would be in the higher quality grades. This would increase profitability of coffee production.
As is the case with many other fruit Yang and Hoffman, Ann. Rev. Plant Physiol. 35:155 (1984)!, plant-produced ethylene plays an important role in the final stages of fruit ripening in coffee. Once coffee fruit reach a certain stage of maturity they can be induced to ripen by the exogenous application of ethylene Crisosto, C. H., P. C. Tausend, M. A. Nagao, L. H. Fuchigami and T. H. H. Chen, J. Haw. Pac. Agri. 3:13-17 (1991). This demonstrates the importance of ethylene for the final stages of fruit ripening in coffee.
Ethylene is synthesized in a two-step reaction from S-adenosylmethionine (SAM). The first step is the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) from SAM by ACC synthase. In most plants this is the rate limiting step. The final step is the conversion of ACC to ethylene which is catalyzed by ACC oxidase (Yang and Hoffman, supra). Inhibition of ethylene biosynthesis by chemical (e.g., silver ions or carbon dioxide) or biotechnological means Oeller et al., Science 254:437 (1991)! inhibits the final stages of ripening. This inhibition is reversible by the application of ethylene.
Accordingly, a strategy for controlling the ripening of coffee plants is to prevent synthesis of specific enzymes in the pathway for ethylene biosynthesis. In one embodiment this invention relates to genetic alteration of coffee plants to eliminate synthesis of ACC synthase; in another, ACC oxidase synthesis is suppressed. In the presently preferred embodiments, synthesis of one or both of these enzymes is suppressed by transforming coffee plants with a DNA sequence that codes on transcription for a messenger RNA (mRNA) that is antisense to the mRNA that codes on expression for the enzyme whose synthesis is to be suppressed. See Oeller et al., Science 254:437 (1991), who reported controlling ripening of tomatoes using a similar strategy.
Recombinant DNA technology has been used to isolate a number of ACC synthase and ACC oxidase genes. However, the genes for ACC synthase and ACC oxidase in coffee have not been identified or sequenced to date.